The detection of chemical processes on a single molecule scale is the ultimate goal of sensitive analytical assays. Derivatives of gramicidin A (gA) show promise as ultra-sensitive transducers for detecting chemical and biochemical analytes due to the high amplification that is inherent to the ion channel activity of these helical peptides. In general, the opening of a single ion channel results in the measurable flux of about 103 to 106 ions per millisecond across lipid membranes. Gramicidin A is a natural ion channel-forming peptide secreted by the bacterium Bacillus brevis. The peptide facilitates a transmembrane flux of monovalent cations upon reversible head-to-head dimerization in lipid bilayers. Gramicidin A is well suited for biosensor applications due to its defined and quantized characteristics of ion channel conductance. For example, the majority of ion channel proteins are reconstituted into bilayers by proteoliposome fusion. In contrast, gA can be dissolved in aqueous solutions and spontaneously incorporates into bilayers. Once in the membrane, gA self-assembles into a functional dimeric nanostructure with characteristic, transient ion flux.
The C-terminus of gA may be derivatized chemically without loss of ion channel activity. Changes in the single-channel conductance, γ, of gA in response to derivatization at the C-terminus may be detected. In general there is low background interference of the ion channel signal detected from non-ionic molecules in solution. For example, an ion channel-based sensor does not rely on optical detection. Accordingly, colored molecules in solution do not interfere provided that they do not influence the passage of monovalent cations through gA pores or alter significantly the ionic strength of the recording buffer. With regards to interference, several characteristics of gA pores are important to consider. for example, only small monovalent cations (e.g., monovalent metal ions or protons) can pass through gA channels. Divalent cations such as Ca2+ are known to block gA channels at concentrations≧100 mM (see Bamberg and Lauger, Membrane Biol., 1977, 35, 351-375). Large concentrations of monovalent cations or divalent cations from analytical samples can therefore be expected to cause interference. Molecules larger than 5 Å cannot pass since gA channels have a diameter of ˜4 Å; these large molecules are not expected to cause significant interference (regardless of whether they are colored or fluorescent) as long as they cannot block gA pores. Therefore, the presence of significant concentrations of monovalent or divalent cations should be accounted for when measuring the conductance of gA derivatives (for instance by calibration or standard addition).
In order to exploit the potentially useful advantages of gA for detection purposes, several groups have explored the use of gA for a number of sensor applications. One of the first descriptions of sensor based on gramicidin A was in 1997 (Cornell et al., Nature 1997, 387, 580-583). Other ion channel platforms such as ion channel proteins (e.g., a-Hemolysin: Bayley and Cremer, Nature 2001, 413, 226-230; Luchian et al., Chem., Int. Ed. 2003, 42, 3766-3771; Luchian et al., Chem., Int. Ed. 2003, 42, 1925-1929; Shin et al., Chem., Int. Ed. 2002, 41, 3707-3709) have provided further demonstrations of the potential advantages of using ion channels as sensors.
The proteins used in previous studies often require genetic engineering and molecular biology expertise and are less amenable to a large user base. Derivatives of gA, for instance, have been used to sense protein-ligand interactions (Cornell et al., Nature 1997, 387, 580-583), redox potential of the electrolyte solution (Antonenko et al., Biochim. Biophys. Acta 2006, 1758, 493-498.) ammonium ions (Nikolelis and Siontorou, Anal. Chem. 1996, 68, 1735-1741.), light (Stankovic et al., Biochim. Biophys. Acta 1991, 1061, 163-170; Banghart eta al., Biochemistry 2006, 45, 15129-15141.) and pH at membrane interfaces (Borisenko et al., Biochim. Biophys. Acta 2002, 1558, 26-33).
Others have demonstrated that charged derivatives of gA can have markedly different conductance values based on the polarity and number of the attached charges (Bamberg et al., Proc. Natl. Acad. Sci. USA, 1978, 37, 2633-2638; Apell et al., Biochim. Biophys. Acta 1979, 552, 369-378). In addition, the functional properties of a chemical analyte (i.e. its reactivity with certain functional groups) can be used to change the chemical properties (i.e., the charge) of these reactive groups covalently attached to the opening of gA. This change in chemical properties can be monitored in situ by using single ion channel recordings (see e.g., FIG. 1; see also Blake et al., Chembiochem 2006, 7, 433-435, 34).
Among the largest limitations of using ion channel-forming peptides such as gramicidin A for development of sensors is the synthetic accessibility of the peptide for readily generating tailored ion channels. Previously described chemical methods for synthesizing C-terminal derivatives of gramicidin A generally include procedures such as esterification (Blake et al., Chembiochem. 2006, 7, 433-435; Apell et al., Membr. Biol. 1977, 31, 171-188; Futaki et al., Bioorg. Med. Chem. 2004, 12, 1343-1350), carbamoylation (Borisenko et al., Biochim. Biophys. Acta 2002, 1558, 26-33), or solid phase synthesis (Roeske et al., Biochim. Biophys. Acta 1989, 982, 223-227). Such procedural limitations represent potential bottlenecks in the development of gramicidin A-based ion channel sensors.
There is a need for derivatives of gA that may be useful as a platform for sensing chemical or biochemical analytes that change the charge of functional groups attached to the entrance of these semi-synthetic nanopores.